Telomeres, the structures present at chromosomal termini, are responsible for the complete replication of the chromosomes and the protection of chromosomes against genomic instability. Most studies have centered on the addition of simple-sequence G+T sequences by the ribonucleoprotein reverse transcriptase, telomerase. Activation of telomere addition, normally through telomerase, is required for oncogenesis. However, alternative recombinational (ALT) pathways are also present in organisms ranging from yeast to humans that can substitute for telomerase. These pathways are poorly understood, leaving a gap in our understanding of telomere addition. Our long-term goal is to understand the mechanisms of both telomeric recombinational interactions, which result in the addition and deletion of telomeric sequences, and the choice between different means of elongation. The objective of this application is to use the budding yeast model system to establish the underlying principles for the regulation of Mre11 in telomere recombination. Our central hypothesis is that characterization of a novel separation-of-function allele of MRE11 wil serve as an initiation point to define the factors, pathways, and telomere states that are associated with telomere recombination. Our rationale is that increasing our knowledge of the mechanism, efficiency, and regulation of telomere recombination will ultimately provide the means for ALT manipulation in human oncogenic cells. These studies will also define the nature of epigenetic heritable states at the telomere, which may change our fundamental view of telomere structure. Recent data that is in press have led to three Specific Aims. First, we want to understand the role of two proteins that act in the same pathway with mre11-A470T: the cell-cycle protein Clb2 and the telomere-specific RFC component, Ctf18. Second, we will use model systems to test whether break-induced replication (BIR) or other mechanisms are involved in the expansion of telomere tracts. Third, we will investigate the heritable epigenetic states that are characteristic of the mre11-A470T allele. The approach is innovative in that it uses an mre11 separation-of-function allele to answer these questions. This proposal is significant because it seeks to increase our understanding of the mechanism and regulation of ALT pathways in both yeast and human systems. Insights from yeast telomere recombination will provide the blueprint for future research on vertebrate ALT cancer-associated pathways and their relationship to epigenetic states. PUBLIC HEALTH RELEVANCE: The relevance of this project to the mission of the NIH is that maintenance of the termini of eukaryotic chromosomes {telomeres} is critical in preventing genomic instability, rearrangement, and oncogenesis. The proposed studies on recombinational pathways of yeast telomere addition, using a unique allele within the DNA repair protein Mre11, will lead to a better understanding of their mechanisms, and of the role of telomere structure in both oncogenesis and aging. !